Characterization of Milled Wood Lignins and Dehydrogenative

Chapter 18

Downloaded by UNIV OF QUEENSLAND on October 16, 2015 | http://pubs.acs.org Publication Date: August 13, 1998 | doi: 10.1021/bk-1998-0697.ch018

Characterization of Milled Wood Lignins and Dehydrogenative Polymerisates from Monolignols by Carbon-13 NMR Spectroscopy Chen-Loung Chen Department of Wood and Paper Science, North Carolina State University, Raleigh, NC 27695-8005

13

C N M R spectroscopy was used to characterize milled wood lignin (MWL) from spruce (Picea glauca) and Zhong-Yan M u (Bischofia polycarpa) as well as dehydrogenative polymerisates (DHPs) from monolignols. The analysis of a quantitative C N M R spectrum of spruce M W L showed that this guaiacyl type lignin had 8-8', 8-5', 5-5' and 8-O-4' linkages which were interpreted as approximating 2, 9, 24 and 53 per 100 C -units, respectively, whereas the frequency of 7-O-4' linkages was less than 3 per 100 C -units. The frequencies of both 5-5' and 8-O-4' linkages were much higher than values previously estimated. The spectrum of the Zhong-Yan Mu M W L showed that it is a guaiacyl-syringyl lignin with a guaiacylpropane-syringylpropane molar ratio of approximately 86:14. The dehydrogenative polymerization of monolignols is discussed on the basis of the components identified among the reaction products. 13

9

9

Earlier work on the characterization of organic compounds by nuclear magnetic resonance (NMR) spectroscopy mainly relied on *H N M R spectroscopy. The major reason for this is that the H nucleus is the most abundant among the nuclei that can be detected by N M R techniques. However, the advent of Fourier transform N M R (FT-NMR) spectroscopic techniques heralded rapid developments in the feasibility of obtaining natural abundance C N M R spectra within reasonable periods of time. Thus, in spite of the low natural abundance of C (1.1 atom %) compared to H (99.98%), C N M R spectroscopy has become as useful as *H N M R spectroscopy for the structural analyses of organic substances. Among the various physical and chemical methods available for characterizing lignin, natural abundance C N M R spectroscopy has been shown to be among the most reliable and comprehensive of the techniques (1-11). It furnishes comprehensive data about the structures of all carbons in lignin, in contrast to other physical and chemical analytical methods which may only supply partial structural information. 1

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©1998 American Chemical Society

In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

l

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256 13

Advantages of C NMR over *H NMR Spectroscopy 1 3

!

There are several advantages of C N M R over H N M R spectroscopy for the structural determination of organic compounds. In a C N M R spectrum, spectral data are obtained from the 'backbone' of the molecule that provides information about the nature of all carbons in the molecule, whether carbonyl, nitrile, quaternary groups, and so forth. The second advantage is that C N M R spectra are not complicated by spin-spin coupling between C nuclei. This is because the probability of having two such nuclei adjacent to each other in the same molecule is low enough that the possibility of C- C coupling can be ignored. Moreover, C N M R spectra are usually obtained with proton decoupling (72), so that only single carbon resonances are observed. The third advantage is that the C N M R chemical shift range for the majority of diamagnetic organic compounds is approximately 240 ppm compared to about 12 ppm for *H N M R spectroscopy. Finally, the signals from samples in solution are normally relatively sharp. A l l these factors imply that there is better resolution and less overlap of the signals in the C N M R spectra of organic compounds. This is of particular value for polymeric natural products such as lignin. 1 3

1 3

1 3

13

13


13

13

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Disadvantages of C NMR over *H NMR Spectroscopy 13

One of the disadvantages of C N M R over *Η N M R spectroscopy is that the peak areas in a C N M R spectrum, obtained with continuous proton decoupling, may not be proportional to the number of the respective C N M R nuclei giving rise to these signals because of the nuclear Overhauser enhancement (nOe) effect and different relaxation times of the various carbons. In order to obtain a quantitative C N M R spectrum for an organic compound, an inverse gated decoupling pulse sequence (where the proton irradiation is switched off during the relaxation delay before the C excitation pulse) must be used to minimize nOe effects while the relaxation delay between successive π/2 pulses must be at least 5 times the longest C longitudinal relaxation time. The other disadvantage is that the C nucleus is approximately 5700 times less sensitive to N M R detection than the Η nucleus because of its relatively low natural isotopic abundance and smaller magnetogyric ratio. 13

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13

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1

Experimental Procedures 13

Preparation of Lignin Samples. To obtain C N M R spectra of naturally occurring lignin, the lignin must be isolated free from other components present in the plant tissues (13). For this purpose, the procedure of Bjorkman (14) is used, in general, to prepare milled wood lignin samples (MWL) from the corresponding woody plant tissues. The milled wood lignin usually contains up to 5% of carbohydrates. Technical lignins obtained from pulping spent liquors also contain appreciable amounts of carbohydrates and fatty acids. Therefore, care must be taken to remove these contaminants as much as possible when purifying both M W L and technical lignin preparations. After purification, the lignin preparations must be dried in a drying pistol at 50°C in vacuo for 24 h using P Ô as drying agent. The dried, purified lignin preparations are then often subjected to elemental (C, H , and O), methoxyl and carbohydrate analyses prior to obtaining their C N M R spectra. 2

5

13

Preparation of Sample Solution. About 300-400 mg of the dried, purified lignin preparation is dissolved in 2 mL of a suitable solvent, with tetramethylsilane (TMS) being added as an internal reference. Insoluble materials, if any, are filtered off with a sintered glass funnel (filter disk 10 mm, i.d., porosity 25-50 mm). The concentration of the sample is typically ca. 15-20% (w/v). The sample solution is then transferred to a 5 mm or 10 mm diameter N M R sample tube, depending upon the probe of the instrument. In general, a C N M R spectrum of a lignin preparation in a suitable solution is obtained with a C N M R spectrometer operating in a Fourier transform (FT) mode at a frequency of more than 50 M H z for the C nucleus. A 13

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solution of the lignin is placed in the probe of the N M R spectrometer, and the H nucleus of the solvent, i.e. either dimethylsulfoxide-d (DMSO-d ) or acetone-d , is used as an internal lock for the spectrometer radio-frequency field. The optimum parameters for the operation of the spectrometer are determined in part by the experimental data obtained. 6

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6

6

1 3

Routine C NMR Spectra. In general, a routine C N M R spectrum of a lignin preparation is recorded at 25-50°C with a pulse width corresponding to a flip angle in the range of 30-60°, a data acquisition time of ca. 0.5-1.0 sec and a pulse delay of ca. 0.5-2 sec, with the number of transient acquisitions being ca. 10,000-20,000.


13

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Quantitative C NMR Spectra. To facilitate effective analysis of a lignin C N M R spectrum, the spectrum must be quantitative as far as peak areas are concerned (7, 8). In this regard the following factor must be considered: the excitation pulse width ; the time required to reestablish the Boltzmann equilibrium state after the rf pulse; elimination of nuclear Overhauser enhancement due to proton decoupling; and the number of transient acquisitions to obtain a reasonable signal-to-noise ratio. In general, a quantitative C N M R spectrum of lignin can be obtained by an inverse gated decoupling (IGD) pulse sequence using a 90° pulse with a data acquisition time of ca. 0.6 sec and pulse delay of ca. 12 sec employing ca. 20,000 transient acquisitions at an operational frequency of more than 50.3 MHz. The quantitative accuracy of the spectrum must be verified, as will be discussed in the following section. Results and Discussion 13

Analysis of the C NMR Spectrum of Milled Wood Lignin (MWL) from Spruce (Picea glauca). Figure 1 shows a quantitative C N M R spectrum of milled wood lignin (MWL) from spruce (Picea glauca). It was recorded with a Bruker W M 250 N M R spectrometer operating at 62.9 MHz for the frequency of the C nuclei and using an I G D pulse sequence and quadrature detection (75). Deuterated dimethylsulfoxide (DMSO-d ) was used as solvent. The spectrum was acquired from a 10 mm o.d. sample tube with a sample concentration of approximately 17.5% (wlv) at 50°C using a 90° pulse corresponding to a 22 msec pulse width, 14,285 Hz sweep width and 11 sec pulse delay with approximately 15,000transient acquisitions. TMS was used as the internal chemical shift reference. DEPT or A P T spectra of the samples may be useful for discriminating between primary and tertiary carbon signals, on the one hand, and secondary and quaternary carbon signals, on the other. The absence of signals in the 110-103 ppm chemical shift range of the spectrum shows clearly that the lignin is a guaiacyl lignin characteristic of those found in softwoods (gymnosperms). 13

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6

Quantitative Nature of the Spectrum. The spectrum shows that the signal at 55.6 ppm, an aromatic methoxyl carbon, integrates at approximately 0.98 carbon per aromatic ring (Tables I and II). This corresponds to a frequency of approximately 0.98 O C H per C -unit for the M W L . The elemental analysis of the M W L gave a methoxyl content of 0.94 per C -unit [C H . 0 (H 0) 0 . 4 4 ^ ^ ) 0 . 9 4 ] M W L (75, 16). Thus, the methoxyl content estimated from the C N M R spectrum deviates by approximately 4% from that obtained by elemental analysis. This is well within the ±5% deviation that can be expected for the C N M R spectroscopic estimate, and thus the C N M R spectrum has been confirmed to be quantitative. 3

9

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9

7

66

2

2 1 3

13

13

Selection of the 160-103 ppm Chemical Shift Range of the Predominantly Aromatic Region as an Internal Standard. In general, the predominantly aromatic region (160-103 ppm) contains an insignificant number of signals originating from contaminants if the M W L preparation is properly purified (16). It is also well established that spruce M W L contains approximately three 4-0-alkylated coniferyl In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


258

η

1

1

1

1

200

j

1

1

1

1

j

150

1

1

1

i

100 *

1~

50 δ in

PP

m

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Figure 1. Quantitative C N M R spectrum of milled wood lignin (MWL) from spruce (Picea glauca) obtained by inverse gated proton decoupling. Solvent: DMSO-d . (Adapted from ref. 75.) 6



259

Table I. Number of Carbons Corresponding to Chemical Shift Regions for the C NMR Spectrum of Spruce (Picea glauca) Milled Wood Lignin (MWL). 13

a b

a

Chemical Shift Range (ppm)

Integral

194-193 192-191 158-154 154-150.5 150.5-148.5 148.5-145.5 145.5-140 140-124 124-103 90-57 57-54.5 54.5-52.5

1.1 0.7 0.8 6.8 6.2 19.8 6.1 27.9 48.4 44.0 18.6 2.4

b

Number of Carbons per Aromatic Ring 0.06 0.04 0.04 0.36 0.33 1.04 0.32 1.47 2.56 2.32 0.98 0.13

Adapted from reference 15. Total integral for 160-100 ppm chemical shift range =116. Thus, the integral for one aromatic carbon = 116/6.12 = 18.95, assuming that spruce M W L contains 3 units of Ar-CH=CH-CHO and 3 units of Ar-CH=CH-CEUOH substructures per 100 C -units. For type of carbon see Table Π. Ar = 3-methoxy-4-hydroxyphenyl. 9

c

0


260 Table II.

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C Chemical Shifts and Signal Assignments for Spruce (Picea glauca) Milled Wood Lignin (MWL). Solvent: DMSO-rf,. a


Signal Number

a

b

0

Chemical Shift (ppm)

Signal Intensity

1

193.4

w

2 3 4

191.6 169.4 166.2

vw w vw

5 6 7

162.3 156.4 152.9

vw vw w

8

152.1

w

9

151.3

w

10 11 12 13

149.4 149.1 146.8 146.6

s s s s

14

145.8

m

15 16

145.0 143.3

m m

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

138.0 134.6 132.4 131.1 129.3 128.0 125.9 122.6 119.9 118.4 115.1 114.7 111.2 110.4 86.6 84.6 83.8 71.8 71.2

w w w m w w w vw m s s s s m vw w m m m

36 37 38 39 40 41

63.2 62.8 60.2 55.6 53.9 53.4

w w s vs vw vw

15

0

Assignment

C=0 in A r - C H = C H - C H O C=0 in A r - C O - C H ( - O A r ) - C C=0 in A r - C H O ester C=0 in R - 0 - C O - C H C=0 in A r - C O O H ; ester C=0 in Ar-CO-OR unknown C-4 in H-units C-3/C-3' in etherified 5-5' units; C-7 in A r - C H = C H - C H O units C-3/C-5 in etherified S-units and B-ring of 4-0-5' units C-4 in etherified G-units with 7-C=0 group C-3 in etherified G-units C-3 in etherified G-units (8-0-4' type) C-4 in etherified G-units C-3 in nonetherified G-units (8-0-4' type) C-4 in nonetherified G-units C-4' in ring B of 8-5' units C-4/C-4' of etherified 5-5' units C-3' in ring Β of 8-5' units C-4/C-4' of nonetherified 5-5' units unknown C - l in etherified G-units C-5/C-5' in etherified 5-5' units C - l in nonetherified G-units C-8 in A r - C H = C H - C H O C-7 and C-8 in A r - C H = C H - C H O H C-5/C-5' in nonetherified 5-5' units C - l and C-6 in A r - C O - C - C units C-6 in G-units C-6 in G-units C-5 in G-units C-5 in G-units C-2 in G-units C-2 in G-units C-8 in S type 8 - 0 ^ ' units (erythro) C-8 in G type 8-0-4' units (threo) C-8 in G type 8-0-4' units (erythro) C-7 in G type 8-0-4' units (erythro) C-7 in G type 8-0-4' units (threo) C-9inGtype 8-8'units C-9 in G type 8-0-4' units w/ 7-C=0 C-9inGtype 8-5', 8-1'units C-9 in G type 8-0-4'units C in A r - O C H , C-8/C-8' in 8-8' units C-8 in 8-5' units 1

2

Adapted from reference 15. vw = very weak; w = weak; m = moderate; s = strong; vs = very strong. H = p-hydroxyphenylpropane; G = guaiacylpropane; S = syringylpropane.


261 aldehyde units and three 4 - 0 -alkylated coniferyl alcohol units per 100 C -units. Thus, the signals in this range embody those from 6.12 aromatic and alkenyl carbons. It follows that the total integral over this range divided by 6.12 is equivalent to one carbon atom. 9

Estimation of Phenylcoumaran (8-5') Units in the Spruce MWL. The 54.552.5 ppm chemical shift range embodies 0.13 carbons/aromatic ring. Consequently the total frequency of 8-5' and 8-8' linkages is approximately 12-14 per 100 C -units. Since the frequency of 8-8' linkages in spruce lignin was estimated to be 2 per 100 C -units by potassium permanganate oxidation (77, 18), the total carbons in the 8-8' units is viewed to approximate 4 per 100 C -units. It follows, therefore, that the total frequency of 8-5' linkages is approximately 9 per 100 C -units and that the total C-8 carbons in the 8-5' units is approximately 8-10 per 100 C -units. This carbon gives rise to the signal at 53.4 ppm (79). Erickson et al. (77) previously gave a frequency of approximately 12 such units per 100 C -units for spruce M W L , on the basis of potassium permanganate oxidation studies. 9

9

9

9


9

9

Nature of Signals in the 194-191 ppm Chemical Shift Region. The chemical shift range around 194 ppm embodies approximately 0.06 carbons per aromatic ring, and may include the -CHO of 4-0-alkylated coniferyl aldehyde groups, and the -C=0 of 4-0-alkylated uncondensed guaiacylpropane units with a 7-carbonyl group. The former were estimated as 3 units per 100 C -units on the basis of the Wiesner reaction (18), and consequently the 4-O-alkylated guaiacylpropane units with a 7-carbonyl group may also approximate 3 in number per 100 Q-units. In addition, the signal at 191 ppm approximates 0.04 carbons per aromatic ring and corresponds to the -CHO of 4-0-alkylated vanillin substructures. Thus, the total number of 4-O-alkylated uncondensed guaiacyl residues with a 7-carbonyl is approximately 7 per 100 C -units. 9

9

Estimation of the Total Biphenyl (5-5') Units in the Spruce M W L . The signals in the 154-150.5 ppm chemical shift range embody approximately 0.36 carbons/aromatic ring. The carbons that could give rise to the resonances in this region are: C-3/C-3' of etherified biphenyl (5-5') substructures and C-3' of halfetherified 5 - 5 ' units (75); C-37C-5' in ring Β of diphenyl ether (4-0-5') substructures; C-4 of 4-O-alkylated uncondensed guaiacyl residues with a 7-carbonyl group (4); C-3/C-5 of 4-0-alkylated, uncondensed syringylpropane units (7, 20); and C-7 of cinnamaldehyde (Ar-CH=CH-CHO) units (7). In general, spruce M W L contains approximately two 4 - 0 - 5 ' substructures ( 18), seven 4-O-alkylated uncondensed guaiacyl residues with a 7-carbonyl group (see Nature of Signals in the 194-191 ppm Chemical Shift Region), three 4—O-alkylated coniferyl aldehyde substructures, one 4-0-alkylated uncondensed syringylpropane unit and three cinnamaldehyde (Ar-CH=CH-CHO) units per 100 C -units. The total number of the C-3/C-3' in etherified 5-5' units and C-3' in half-etherified 5-5' units is therefore approximately 0.20/aromatic ring [= 0.36 - 0.04 - 0.07 - 0.02 - 0.03]. This implies that approximately 19-21 guaiacylpropane units per 100 units are involved in etherified and half-etherified 5-5' units. Furthermore, the 145.5-140 ppm chemical shift range embodies approximately 0.32 carbons/aromatic ring. The carbons giving rise to these resonances are the C-4/ C-4' in the 5-5' units, regardless of whether etherified or not (75), and the C-3' in 8-5' units (27). As discussed previously (see Estimation of Phenylcoumaran (8-5') Units in the spruce MWL), the total C-8 of 8-5' units is estimated from the 54.5-52.5 ppm chemical shift range to be approximately 8-10 per 100 C -units. Thus, even considering the possible extent of experimental error, the spruce M W L contains approximately 22-24 guaiacylpropane residues per 100 C -units that are involved in 5-5' linkages, i.e. the total frequency of 5-5' linkages is approximately 12 per 100 C units. This value is slightly higher than the 10 per 100 Q-unit estimated using the potassium permanganate oxidation method (77, 18). 9

9

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9


262 13

A further inspection of the C N M R spectrum of spruce M W L reveals that fully etherified 5-5' units predominate over those which are partially (half) or nonetherified. This is evidenced by the presence of signals in the 145-144 ppm range and the absence of any resonances between 143 and 142 ppm (75). The former correspond to C-4/C-4' in etherified 5-5' substructures and C-4 in the etherified ring of partially etherified 5-5' linkages, while the latter would correspond to C-4 in the phenolic rings of half- or non-etherified 5-5' units. The C-3' in 8-5' units would also give rise to a signal in the range between 144 and 143 ppm (27). Thus, of the 23-25 guaiacylpropanes per 100 C -units involved in 5-5' linkages, approximately 19-21 are present as fully or half- etherified units. The remainder, approximately 4-6, are half or non-phenolic 5-5' units. It is of interest to note that the fully etherified 5-5' units may occur in lignin in the form of dibenzo-2H,3H-l,4-dioxicin structures (22, 23) that may undergo mechanolysis during the ball milling of spruce wood in the process of milled wood lignin isolation to give partially half etherified 4 ' - 0 - 8 " linked 5-5' units (24).


9

Estimation of 8-Aryl Ether ( 8-0-4') Units in Spruce MWL. The 150.5-148.5 ppm chemical shift range embodies 0.33 carbons/aromatic ring. The signals in this range correspond to the C-3 of etherified guaiacyl groups (20). The frequency of and 8-0-4' linkages can be estimated as approximately 32-34 per 100 C units. This does not include 7 - 0 - 4 ' and 8 - 0 u n i t s having a 5-5' linkage as an aryl ether moiety, such as the dibenzodioxicin and half-etherified 4 ' - 0 - 8 " linked 5-5' units. Assuming that the fully and half-etherified 5-5' units contributing to 1-0-4' and 8-0-4' linkages are in dibenzodioxicin and (half-etherified) 4 ' - 0 - 8 ' linked 5-5' substructures, then the total frequency of 1-0-4' and 8-0-4' linkages would be approximately 53 [= 33 + 20] per 100 C -units. Since the frequency of 7-0-4' units is estimated to be approximately 8 per 100 C -units (77, 18, 25), the frequency of 8-0-4' linkages is estimated at 44-46 per 100 C -units. Very recently, Ede and Kilpelâinen searched for 7-0-4' units in the MWL's from a number of softwood and hardwood species employing 2D N M R spectroscopic methods such as the H O H A H A and H M Q C techniques (26). The results indicate that, if 1-0-4' units are present in MWL's, they are present at a level below the detection limit of the techniques, that is less than 0.3 per 100 C -units. The implications of this finding are twofold: the frequency of the 8-0-4' linkages in softwood lignin is approximately 52-54 per 100 C -units, which is much higher than the values previously estimated; and the number of dibenzo-2H,3H-l,4-dioxicin structures present in softwood lignin is insignificant. 9

9

9

9

9

9

Estimation of Degree of 'Condensation'. Softwood lignins contain several 'condensed' aromatic structures such as the biphenyl (5-5') and phenylcoumaran (8-5') linkages. If they did not contain 'condensed' aromatic structures, then the signals in the 124-103 ppm chemical shift range should embody 3 carbons per aromatic ring. The deficiency in the number of carbons in this chemical shift range must then be due to the presence of 'condensed' aromatic structures. Thus, the total number of 'condensed' aromatic units and the degree of 'condensation', can be estimated by subtracting from the total number of carbons per aromatic ring estimated from the 124-103 ppm chemical shift range. Thus, the degree of 'condensation' for the spruce M W L was approximately 0.44 [= 3 - 2.56] per aromatic ring, i.e. 44 per 100 C -units. 9

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Analysis of the C NMR Spectrum of Milled Wood Lignin (MWL) from ZhongYang Mu (Bischofia polycarpa). Zhong-Yang M u (Bischofia polycarpa; Euphorbiaceae) is a hardwood-timber species widely distributed in southeastern China. Figure 2 shows the quantitative C N M R spectrum of the M W L from this wood species acquired using inverse gated proton decoupling (27). The quantitative nature of the spectrum is confirmed by the fact that signal 29 (aromatic methoxyl carbons) in the spectrum integrates to approximately 1.16 carbons per phenyl group In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998. 13

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Figure 2. Quantitative C N M R spectrum of milled wood lignin (MWL) from Zhong-Yang M u (Bischofia polycarpa) obtained by inverse gated proton decoupling. Solvent: DMSO-d . (Adapted from ref. 27.)


264 (Tables III and IV). This corresponds to a methoxyl content of approximately 1.16 per C -unit. The elemental analysis of the M W L gave a methoxyl content of 1.13 per C -unit [C H 4 0 (H 0)o.92(OCH3) ]. Thus, the methoxyl content of the M W L estimated from the C N M R spectrum differs by less than 3% from the value obtained by elemental analysis. This is within a 5% deviation from the latter value, the limit of error that can be expected for the C N M R spectroscopic estimation. 9

9

9

6

9

2

2

1J3

1 3

13

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CH, C H and C H C NMR Subspectra for B. polycarpa MWL. Figure 3 shows the C H , C H and C H C N M R subspectra of the B. polycarpa M W L , edited by the DEPT sequence (28-31), while Figure 4 depicts the quaternary C N M R subspectrum of the M W L obtained by subtracting the DEPT edited C H , C H and C H subspectra from the full inverse gated decoupled C N M R spectrum (27). In Table III, the quantitative C N M R spectrum of the M W L is divided into several spectral regions on the basis of the DEPT edited subspectra. Table IV shows the assignments of signals which were deduced from previously published data (7-9, 15,16,19, 20, 21). 2

3

13

2

3

1 3

2

3

13


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Verification of the Presence of Carbohydrates. Carbohydrate analysis of the B. polycarpa M W L showed that it possessed a carbohydrate content of approximately 6.2%. Indeed, the inversed gated decoupled C N M R spectrum of the M W L (Figure 2 and Table IV) reveals that the M W L contains a substantial amount of carbohydrates as evidenced by the presence of signals 4, 20, 24 and 32 at 169.6, 101.6, 76-73 and 20.9 ppm corresponding to Ο-acetyl C=0, C - l and C-2 of xylan, C-3 and C-4 of xylan, and Ο-acetyl C H , respectively. Thus, the C N M R spectral data support the results of the carbohydrate analysis of the M W L (27). 13

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3

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Guaiacyl-Syringyl Nature of B. polycarpa MWL. The quantitative C N M R spectrum of the B. polycarpa M W L (Figure 2) further shows that its lignin is of a guaiacyl-syringyl type. The presence of guaiacyl-propane units is evidenced by signals 14, 15 and 16 at 119.6, 115.8 and 112.2 ppm corresponding to C-6, C-5 and C-2, respectively, of the guaiacyl aromatic ring. The presence of syringylpropane units is revealed in signals 18 and 19 at 104.9 and 103.7 ppm, both corresponding to C-2/C-6 of the syringyl aromatic ring, in addition to signal 6 at 152.6-152.3 ppm corresponding to C-3/C-5 of 4-O-alkylated syringyl residues (20). Predominance of 8-Aryl Ether (8-0-4') Linkages. In contrast to the rather weak signal 9 at 145.6 ppm corresponding to C-4 of the guaiacyl ring, signals 7 and 8 at 149.9-149.4 ppm and 147.7-147.2 ppm are very strong (Figure 2). Since signals 7 and 8 correspond to C-3 and C-4 of 4-O-alkylated guaiacyl rings (20), these moieties must be present in the lignins predominantly as 4-0-alky 1 ethers. Moreover, they seem to be present in the lignin in the form of 8 - 0 ^ ' units because of the presence of relatively strong signals 22, 25 and 28 at 84.6, 71.8 and 60.2 ppm, respectively. These signals correspond to C-8, C-7 and C-9/8-0-4' linkages, respectively (19). The presence of syringylpropane structures is also revealed in signals 18 and 19 at 104.9 and 103.7 ppm, both corresponding to the C-2/C-6 of syringyl rings, in addition to signal 6 at 152.6-152.3 corresponding to C-3/C-5 of 4-O-alkylated syringyl residues (20). In addition to signals 25 and 26, the presence of signal 21 at 87.0 ppm further indicates that 4-O-alkylated syringylpropane units are also present in the lignins mostly in the form of 8-0^1' linkages. Signals 30 and 31 at 53.8 and 53.4 ppm indicate the presence of 8-8'and 8-5'substructures in the lignin sample. Selection of Aromatic Region (160-103 ppm) as an Internal Standard. The aromatic region (160-103 ppm) in the inversed proton decoupled C N M R spectrum, was again chosen as an internal standard to analyze the spectra quantitatively, since there are no signals from carbohydrate contaminants. The DEPT edited C H subspectrum (Figure 3a) of the B. polycarpa M W L indicates the presence of cinnamaldehyde- and cinnamyl-alcohol-type structures as revealed in signals 1, 5 and 13



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Table III. Number of Carbons Corresponding to Chemical Shift Ranges for C NMR Spectrum of Zhong-Yang Mu (Bischofiapolycarpa) Milled Wood Lignin (MWL). a

Spectral Region d

Aromatic quaternary C Aromatic tertiary C Syringyl C-2/C-6 Oxygenated aliphatic C Aromatic methoxyl C C-8 in 8-8' & 8-5' a b

e

Chemical Shift Range (ppm)

Integral

156-128 128-103 108-103 90.0-57.5 57.5-54.5 54.5-52.5

33.0 23.0 3.3 28.6 10.6 0.8

Number of Carbons Per Aromatic Ring 3.61 2.51 0.36 3.13 1.16 0.09

Adapted from reference 27. Total integral for 160-103 ppm chemical shift range = 56; thus, the integral for one aromatic carbon = 56/6.12 = 9.15, assuming that the Zhong-Yang M u M W L contains 3 Ar-CH=CH-CHO and 3 A r - C H = C H - C H O H substructures per per 100 C -units. For type of carbon see Table IV. Including 0.12 vinyl carbons /aromatic ring. Consisting of carbons in lignin sidechains and carbohydrates. 2

9

c d e

0


266 1 3

T a b l e I V . C C h e m i c a l Shifts a n d Signal Assignments for Z h o n g - Y a n g M u (Bischofiapolycarpa) M i l l e d W o o d L i g n i n ( M W L ) . Solvent: D M S O - r f , .


a

Signal Number

Chemical Shift (δ)

Intensity

1

194.0

vw

2 3 4 5 6

191.6 172.0 169.6 152.9 152.9152.3

vw vw w vw s

7

149.4149.2 147.7147.2

s

8

a

b

c

0

Assignment

15

s

9 10

145.6 143.2

w m

11

135.6

s

12

135.0131.0

m

13

m

14 15 16 17

130.0129.0 119.0 115.8 112.2 106.5

s s s w

18

104.7

m

19

103.7

m

20 21 22 23 24

vw m s m w

25 26

101.6 87.0 84.6 81.2 76.073.0 72.2 71.6

27

62.9

s

28 29 30 31 32

60.2 55.8 53.8 53.4 20.4

s vs w vw s

C=0 in Ar-CH=CH-CHO units; C=0 in Ar-CO-CH(-OAr) - C - units C=0 in A r - C H O units C=0 in -COOH of aliphatic acid units C=0 in O-acetyl groups of xylan units C-7 in Ar-CH=CH-CHO units C-3/C-3' in etherified 5-5' units; C-3/C-5 in etherified S-units and Β-ring of 4-0-5' units C-3 in etherified G-units C-3 in etherified G-units (8-0-4' type); C-3 in nonetherified G-units; C-4 in etherified G-units; C-3/C-5 in nonetherified S-units C-4 in etherified G-units C-4' in B-ring of 8-5' units; C-4/C-4' in nonetherified 5-5' units; C-3' in B-ring of 8-5' units; C-4/C-4' in nonetherified 5-5' units; C-l in etherified G-units; C-4 in etherified S-units C-l in nonetherified G- and S-units; C-5/C-5' in etherified 5-5" units; C - l in nonetherified G-units C-8 in Ar-CH=CH-CHO units; C-7 and C-8 in Ar-CH=CH-CH OH units C-6 in G-units C-5 in G-units C-2 in G-units C-2/C-6 in etherified and nonetherified S-units with 7-C=0 group C-2/C-6 in etherified and nonetherified S-units C-2/C-6 in etherified and nonetherified S-units and S-type 8-8' units C - l in xylan C-8 in S-type 8 - 6 M (erythro) C-8 in G-type 8-0-4' units (erythro) unknown C-2/C-3/C-4 in xylan units 2

s w

r

C-7 in G-type 8-0-4'units (erythro) C-9 in G- and S-type 8-0-4'units (both threo); C-9 in G- and S-type 8-8' units C-9 in G- and S-type 8-5' and 8 - Γ units C-9 in G- and S-type 8-0-4' units with a 7-C=Q; C-5 in xylan C-9 in G- and S-type 8-0-4' units C in Ar-OCH units C-8/C-8' in G- and S-type 8-8' units C-8 in G- and S-type 8-5' units C in C H of O-acetyl groups of xylan 3

3

Adapted from reference 27. vw = very weak; w = weak; m = moderate; s = strong; vs = very strong. G = guaiacylpropane; S = syringylpropane.



1

2

3

1

1

1 1

150

1

13

1

1

6

1 1

100

1

Γ

π

ι 50

2

I

ι

DMSO-c/

6

ι

6

Figure 3. DEPT-edited C N M R subspectra of milled wood lignin (MWL) from Zhong-Yang M u (Bischofia polycarpa): (A) C H , (B) C H , (C) C H subspectra, and (D) overall spectrum obtained by inverse gated proton decoupling. Solvent: D M S O - J . (Adapted from ref. 27.)

CH

(A)

200

—]

CH

(B)

1 2

CH

(C)

KJ

DMSO-

Characterization of Milled Wood Lignins and Dehydrogenative

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